The invention relates to compositions and methods for the treatment of cancer.
Extracellular signals received at transmembrane receptors are relayed into the cells by the signal transduction pathways (Pelech et al., Science 257:1335 (1992)) which have been implicated in a wide array of physiological processes such as: induction of cell proliferation, differentiation or apoptosis (Davis et al., J. Biol. Chem. 268:14553 (1993)). The Mitogen Activated Protein Kinase (MAPK) cascade is a major signaling system by which cells transduce extracellular cues into intracellular responses (Nishida et al., Trends Biochem. Sci. 18:128 (1993); Blumer et al., Trends Biochem. Sci. 19:236 (1994)). Many steps of this cascade are conserved, and homologous for MAP kinases have been discovered in different species.
In mammalian cells, the Extracellular-Signal-Regulated Kinases (ERKs), ERK-1 and ERK-2 are the archetypal and best-studied members of the MAPK family, which all have the unique feature of being activated by phosphorylation on threonine and tyrosine residues by an upstream dual specificity kinase (Posada et al., Science 255:212 (1992); Biggs III et al., Proc. Natl. Acad. Sci. USA 89:6295 (1992); Gamer et al., Genes Dev. 6:1280 (1992)).
Recent studies have identified an additional subgroup of MAPKs, known as c-Jun NH2-terminal kinases 1 and 2 (JNK-1 and JNK-2), that have different substrate specificities and are regulated by different stimuli (Hibi et al., Genes Dev. 7:2135 (1993)). JNKs are members of the class of stress-activated protein kinases (SPKs). JNKs have been shown to be activated by treatment of cells with UV radiation, pro-inflammatory cytokines and environmental stress (Derijard et al., Cell 1025 (1994)). The activated JNK binds to the amino terminus of the c-Jun protein and increases the protein""s transcriptional activity by phosphorylating it at ser63 and ser73 (Adler et al., Proc. Natl. Acad. Sci. USA 89:5341 (1992); Kwok et al., Nature 370:223 (1994)).
Analysis of the deduced primary sequence of the JNKs indicates that they are distantly related to ERKs (Davis, Trends Biochem. Sci. 19:470 (1994)). Both ERKs and JNKs are phosphorylated on Tyr and Thr in response to external stimuli resulting in their activation (Davis, Trends Biochem. Sci. 19:470 (1994)). The phosphorylation (Thr and Tyr) sites, which play a critical role in their activation are conserved between ERKs and JNKs (Davis, Trends Biochem. Sci. 19:470 (1994)). However, these sites of phosphorylation are located within distinct dual phosphorylation motifs: Thr-Pro-Tyr (JNK) and Thr-Glu-Tyr (ERK). Phosphorylation of MAPKs and JNKs by an external signal often involves the activation of protein tyrosine kinases (PTKs) (Gille et al., Nature 358:414 (1992)), which constitute a large family of proteins encompassing several growth factor receptors and other signal transducing molecules.
Protein tyrosine kinases are enzymes which catalyze a well defined chemical reaction: the phosphorylation of a tyrosine residue (Hunter et al., Annu Rev Biochem 54:897 (1985)). Receptor tyrosine kinases in particular are attractive targets for drug design since blockers for the substrate domain of these kinases is likely to yield an effective and selective antiproliferative agent. The potential use of protein tyrosine kinase blockers as antiproliferative agents was recognized as early as 1981, when quercetin was suggested as a PTK blocker (Graziani et al., Eur. J. Biochem. 135:583-589 (1983)).
The best understood MAPK pathway involves extracellular signal-regulated kinases which constitute the Ras/Raf/MEK/ERK kinase cascade (Boudewijn et al., Trends Biochem. Sci. 20, 18 (1995)). Once this pathway is activated by different stimuli, MAPK phosphorylates a variety of proteins including several transcription factors which translocate into the nucleus and activate gene transcription. Negative regulation of this pathway could arrest the cascade of these events.
What are needed are new anticancer chemotherapeutic agents which target receptor tyrosine kinases and which arrest the Ras/Raf/MEK/ERK kinase cascade. Oncoproteins in general, and signal transducing proteins in particular, are likely to be more selective targets for chemotherapy because they represent a subclass of proteins whose activities are essential for cell proliferation, and because their activities are greatly amplified in proliferative diseases.
What is also needed are new anticancer therapeutics which are highly selective in the killing of tumor cells, but not normal cells.
It is an object of the invention to provide compounds, compositions and methods for the treatment of cancer and other proliferative diseases. The biologically active compounds are in the form of (E)-styryl benzylsulfones.
It is an object of the invention to provide compounds which are highly selective in killing tumor cells but not normal cells.
It is a further object of the invention to provide novel polymers prepared by polymerization of (E)-styryl benzylsulfones.
It is a further object of the invention to provide intermediates useful for the preparation of compounds having anticancer activity. The intermediates comprise (E)-styryl benzylsulfonyl acetic acids.
According to one embodiment of the invention, novel compounds are provided according to formula I: 
wherein:
R1, R2, R3, and R4 are independently selected from the group consisting of hydrogen; fluoro; chloro; bromo; C1-C6 alkyl; C1-C6 alkoxy; nitro; cyano; and trifluoromethyl;
with the proviso that
(a) R1, R2, R3, and R4 may not all be hydrogen;
(b) when R1, R2, and R3 are hydrogen, then R4 may not be:
(i) 2- or 4-chloro or 4-fluoro;
(ii) 2-nitro, 3-nitro or 4-nitro;
(iii) 4-methoxy or 4-ethoxy; or
(iv) 4-methyl;
(c) when R1 and R3 are hydrogen and R2 is 4-chloro, then R4 may not be 4-chloro, 4-fluoro, 4-bromo, 4-nitro, 4-isopropyl or 4-ethoxy;
(d) when R1 and R3 are hydrogen and R2 is 4-fluoro, then R4 may not be 4-fluoro, 4-bromo, or 4-chloro
(e) when R1 and R3 are hydrogen and R2 is 4-nitro, then R4 may not be 4-chloro, 4-nitro, 4-bromo, 4-fluoro, 4-methyl, or 4-methoxy;
(f) when R1 and R3 are hydrogen and R2 is 4-methyl, R4 may not be 4-chloro, 4-bromo, 4-fluoro, 4-methyl or 2-chloro;
(g) when R1 and R3 are hydrogen and R2 is 4-bromo, then R4 may not be 4-fluoro, 4-bromo or 4-chloro;
(h) when R1 and R2 are hydrogen, then R3 and R4 may not be 2,4-dichloro, 2,3-dimethoxy or 3,4-dimethoxy;
(i) when R1 is hydrogen, then R2, R3 and R4 may not all be fluoro; and
(j) when R1 is hydrogen and R3 is 2-fluoro, then R2 and R4 may not both be selected from the group consisting of 4-chloro, 4-bromo, and 4-fluoro.
According to a preferred embodiment of the invention, novel compounds are provided according to formula I wherein R1, R2, R3, and R4 are independently selected from the group consisting of hydrogen, chloro, fluoro, bromo, nitro, cyano and trifluoromethyl. According to a more preferred embodiment, R1, R2, R3, and R4 are independently selected from the group consisting of hydrogen, chloro, fluoro and bromo; most preferably hydrogen, chloro and fluoro.
In a further preferred embodiment, novel compounds are provided according to formula I wherein (1) at least one of R1 and R2 is other than hydrogen and is located at the 2-, 3- and/or 4-position of the phenyl ring to which it is attached, and is preferably selected from chloro and fluoro, most preferably chloro; and/or (2) wherein at least one of R3 and R4 is other than hydrogen and is located at the 2- and/or 4-position of the phenyl ring to which it is attached, and is preferably selected from chloro and fluoro. In other preferred embodiments wherein at least one of R1 and R2 is other than hydrogen, and at least one of R3 and R4 is other than hydrogen, (i) R2 is 4-halogen or 4-cyano, and R4 is 4-nitro; or (ii) R2 is 4-C1-C6 alkoxy, and R4 is 4-nitro or 4-halogen. R1 and R3 are preferably hydrogen in these embodiments.
In another embodiment of the invention, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and one or more compounds of formula II: 
wherein
R1, R2, R3, and R4 are independently selected from the group consisting of hydrogen; fluoro; chloro; bromo; C1-C6 alkyl; C1-C6 alkoxy; nitro; cyano; and trifluoromethyl;
with the proviso that
(a) R1, R2, and R3 are not all hydrogen when R4 is 2-chloro or 4-chloro;
(b) when R1 and R3 are hydrogen and R2 is 4-bromo or 4-chloro, then R4 may not be 4-chloro, 4-fluoro or 4-bromo;
(c) when R1 and R3 are hydrogen and R2 is 4-fluoro, R4 may not be 4-fluoro or 4-bromo;
(d) when R1 is hydrogen, and R4 is 2-fluoro, then R2 and R3 may not be 4-fluoro;
(e) when R1 is hydrogen and R3 is 4-hydrogen, 4-chloro, 4-bromo, 4-methyl or 4-methoxy, and R4 is 2-hydrogen, 2-chloro or 2-fluoro; then R2 may not be 4-hydrogen, 4-chloro, 4-fluoro or 4-bromo.
According to a related invention, novel compounds are provided according to formula III: 
wherein
R1, R2, R3, and R4 are independently selected from the group consisting of hydrogen; fluoro; chloro; bromo; iodo; C1-C6 alkyl; C1-C6 alkoxy; nitro; cyano; and trifluoromethyl;
provided at least one of R1, R2, R3, and R4 is iodo.
According to a preferred embodiment, at least one of R1 and R2 in formula III is other than hydrogen and is located at the 2- or 4-position of the phenyl ring to which it is attached; and at least one of R3 and R4 is other than hydrogen and is located at the 2- or 4-position of the phenyl ring to which it is attached. According to a further preferred embodiment, R2 and R4 in formula III are hydrogen, and R1 and R3 are located at the 4-position of the respective phenyl rings to which they are attached. According to a further preferred embodiment, one of R1 or R3 is selected from the group consisting of chloro, fluoro, bromo and nitro, the other of R1 or R3 being iodo.
A pharmaceutical composition is also provided comprising a pharmaceutically acceptable carrier and one or more compounds of formula III above, wherein R1, R2, R3, and R4 are defined as above for formula III.
Where R1, R2, R3 or R4 is an alkyl or alkoxy group in any compound of formulae I, II or III, the carbon chain may be branched or straight, with straight being preferred. Preferably, the alkyl and alkoxy groups comprise C1-C3 alkyl and C1-C4 alkoxy, most preferably methyl and methoxy.
According to another embodiment of the invention, a method of treating an individual for a proliferative disorder, particularly cancer, is provided, comprising administering to said individual an effective amount of a compound according to formula II or III, alone or in combination with a pharmaceutically acceptable carrier.
In another embodiment of the invention, a method of inhibiting growth of tumor cells in an individual afflicted with cancer is provided comprising administering to said individual an effective amount of a compound according to formula II or III, alone or in combination with a pharmaceutically acceptable carrier.
In another embodiment, a method of inducing apoptosis of cancer cells, more preferably tumor cells, in an individual afflicted with cancer is provided, comprising administering to said individual an effective amount of a compound according to formula II or III, alone or in combination with a pharmaceutically acceptable carrier.
In yet another embodiment of the present invention, benzyl sulfones having the structural formula II or III, may be utilized as monomers in the synthesis of a new class of polymers having pendant benzylsulfone groups.
The present invention also provides a series of substituted benzylsulfonyl acetic acid compounds having structural formula V, below. The substituted benzylsulfonyl acetic acid compounds are useful as intermediates in the synthesis of novel (E)-styryl benzylsulfone compounds of formula I, according to Method A, below.
According to the present invention, certain (E)-styryl benzylsulfone derivatives selectively kill various tumor cell types without killing normal cells. Without wishing to be bound by any theory, it is believed that the compounds affect the MAPK signal transduction pathway, thereby affecting tumor cell growth and viability. This cell growth inhibition is associated with regulation of the ERK and JNK types of MAPK. Without wishing to be bound by any theory, the styryl sulfones of the present invention may block the phosphorylating capacity of ERK-2.
The compounds of the invention have been shown to inhibit the proliferation of tumor cells by inducing cell death. The compounds are believed effective against a broad range of tumor types, including but not limited to the following: breast, prostate, ovarian, lung, colorectal, brain (i.e, glioma) and renal. The compounds are also believed effective against leukemic cells. The compounds do not kill normal cells in concentrations at which tumor cells are killed.
The compounds are also useful in the treatment of non-cancer proliferative disorders, including but not limited to the following: hemangiomatosis in new born, secondary progressive multiple sclerosis, chronic progressive myelodegenerative disease, neurofibromatosis, ganlioneuromatosis, keloid formation, Pagets Disease of the bone, fibrocystic disease of the breast, Peronies and Duputren""s fibrosis, restenosis and cirrhosis.
Treatment of this broad range of tumor cells with the styryl benzylsulfone compounds of the invention leads to inhibition of cell proliferation and induction of apoptotic cell death. In breast tumors, the effect is observed for estrogen receptor (ER) positive as well as estrogen receptor negative cells.
Tumor cells treated with the compounds of the invention accumulate in the G2/M phase of the cell cycle. As the cells exit the G2/M phase, they appear to undergo apoptosis. Treatment of normal cells with the styryl sulfones does not result in apoptosis.
The styryl benzylsulfones are characterized by cis-trans isomerism resulting from the presence of one or more double bonds. The compounds are named according to the Cahn-Ingold-Prelog system, the IUPAC 1974 Recommendations, Section E: Stereochemistry, in Nomenclature of Organic Chemistry, John Wiley and Sons, Inc., New York, N.Y., 4th ed., 1992, p. 127-138. Stearic relations around a double bond are designated as xe2x80x9cZxe2x80x9d or xe2x80x9cExe2x80x9d.
(E)-styryl benzylsulfones are prepared by Knoevenagel condensation of aromatic aldehydes with benzylsulfonyl acetic acids. The procedure is described by Reddy et al., Acta. Chim. Hung. 115:269 (1984); Reddy et al., Sulfur Letters 13:83 (1991); Reddy et al., Synthesis 322 (1984); and Reddy et al., Sulfur Letters 7:43 (1987), the entire disclosures of which are incorporated herein by reference.
The (E)-styryl benzylsulfones can be prepared according to either of the following Methods A and B: 
A benzyl thioacetic acid V formed by the reaction of sodium thioglycollate and a benzyl chloride IV. The benzyl thioacetic acid V is oxidized with 30% hydrogen peroxide to give a corresponding benzylsulfonyl acetic acid VI. Condensation of VI with an aromatic aldehyde VII via a Knoevenagel reaction in the presence of benzylamine and glacial acetic acid yields the (E)-styryl benzylsulfone I, II or III.
A benzylthioacetic acid V is formed by the reaction of the appropriate sodium benzylthiolate VIII with chloroacetic acid. Oxidation of V to the corresponding benzylsulfonyl acetic acid VI and subsequent Knoevenagel condensation with aldehyde VII is carried out as in Method A.
Substituted benzylsulfonyl acetic acid compounds Va, Vb, Vc, and Vd according to formula V were prepared by reacting the corresponding benzyl chloride with thioglycollic acid under basic conditions (Method A). These compounds are novel intermediates.
(E)-Styryl benzylsulfones may be utilized as monomers in the synthesis of polymers X having pendant aryl and benzylsulfone groups. The polymerization of styryl benzylsulfones defined according to formula IX below into formula X polymers is accomplished by heating the formula IX compound above 250xc2x0 C. in the presence of a free radical initiator. The initiator may comprise benzoyl peroxide, for example: 
The degree of polymerization in the polymer of formula X, xe2x80x9cxxe2x80x9d, may range from about 10 to about 150, providing an oligomer or polymer of from 5,000 to 50,000 daltons. Other degrees of polymerization are also contemplated. R1, R2, R3, and R4 in the monomer of formula IX, and in the polymer of formula X, are independently selected from the group consisting of hydrogen; halogen, i.e., fluoro, chloro, bromo and iodo, most preferably fluoro, chloro and bromo; C1-C6 alkyl; C1-C6 alkoxy; nitro; cyano; and trifluoromethyl.
The (E)-styryl benzylsulfones may be derivatized with a chemical group to permit conjugation to a carrier molecule, for the purpose of raising antibodies to the styryl sulfones. Suitable derivatizing chemistries are well-known to those skilled in the art. Preferably, the derivative comprises a carboxylic acid derivative. The carrier may comprise any molecule sufficiently large to be capable of generating an immune response in an appropriate host animal. One such preferred carrier is keyhole limpet haemocyanin (KLH).
The (E)-styryl benzylsulfones of the invention may be administered in the form of a pharmaceutical composition, in combination with a pharmaceutically acceptable carrier. The active ingredient in such formulations may comprise from 0.1 to 99.99 weight percent. By xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d is meant any carrier, diluent or excipient which is compatible with the other ingredients of the formulation and to deleterious to the recipient.
The compounds of the invention may be administered to individuals (mammals, including animals and humans) afflicted with cancer. The compounds are also useful in the treatment of non-cancer proliferative disorders, that is, proliferative disorders which are characterized by benign indications. Such disorders may also be known as xe2x80x9ccytoproliferativexe2x80x9d or xe2x80x9chyperproliferativexe2x80x9d in that cells are made by the body at an atypically elevated rate. Such disorders include, but are not limited to, the following: hemangiomatosis in new born, secondary progressive multiple sclerosis, chronic progressive myelodegenerative disease, neurofibromatosis, ganglioneuromatosis, keloid formation, Pagets Disease of the bone, fibrocystic disease of the breast, Peronies and Duputren""s fibrosis, restenosis and cirrhosis.
The compounds may be administered by any route, including oral and parenteral administration. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravaginal, topical or subcutaneous administration. The active agent is preferably administered with a pharmaceutically acceptable carrier selected on the basis of the selected route of administration and standard pharmaceutical practice.
The active agent may be formulated into dosage forms according to standard practices in the field of pharmaceutical preparations. See Gennaro Alphonso, ed., Remington""s Pharmaceutical Sciences, 18th Ed., (1990) Mack Publishing Co., Easton, Pa. Suitable dosage forms may comprise, for example, tablets, capsules, solutions, parenteral solutions, troches, suppositories, or suspensions.
For parenteral administration, the active agent may be mixed with a suitable carrier or diluent such as water, an oil (particularly a vegetable oil), ethanol, saline solution, aqueous dextrose (glucose) and related sugar solutions, glycerol, or a glycol such as propylene glycol or polyethylene glycol. Solutions for parenteral administration preferably contain a water soluble salt of the active agent. Stabilizing agents, antioxidizing agents and preservatives may also be added. Suitable antioxidizing agents include sulfite, ascorbic acid, citric acid and its salts, and sodium EDTA. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol. The composition for parenteral administration may take the form of an aqueous or nonaqueous solution, dispersion, suspension or emulsion.
For oral administration, the active agent may be combined with one or more solid inactive ingredients for the preparation of tablets, capsules, pills, powders, granules or other suitable oral dosage forms. For example, the active agent may be combined with at least one excipient such as fillers, binders, humectants, disintegrating agents, solution retarders, absorption accelerators, wetting agents absorbents or lubricating agents. According to one tablet embodiment, the active agent may be combined with carboxymethylcellulose calcium, magnesium stearate, mannitol and starch, and then formed into tablets by conventional tableting methods.
The specific dose of compound according to the invention to obtain therapeutic benefit will, of course, be determined by the particular circumstances of the individual patient including, the size, weight, age and sex of the patient, the nature and stage of the disease, the aggressiveness of the disease, and the route of administration. For example, a daily dosage of from about 0.05 to about 50 mg/kg/day may be utilized. Higher or lower doses are also contemplated.